It is well established that activated lymphoid cells elaborate a variety of soluble factors (called "lymphokines") participating in the regulation of the immune response [reviewed by Altman et al. (1)]. The profile of factors reported include migration inhibitory factor (2,3), lymphotoxin (4,5), transfer factor (6,7), lymphocyte proliferation inhibitory factor (8,9) and interferon (10,11). Despite the overwhelming number of published studies on lymphokines with immunoregulating activities, the function-structural relationship of these molecules is still poorly understood.
Progress in the study of lymphokines has been strongly impeded by two major limitations. Firstly, due to the heterogenicity of the cell populations used to prepare conventional lymphokines, it is extremely difficult to assign a given biological activity to a distinct molecule. Secondly, the amounts of a given lymphokine in conventional preparations are usually too small to allow a precise biochemical analysis of the biologically active molecules. Moreover, since lymphocytes have a finite and usually short lifetime in culture, it is impossible to obtain repeatedly homogenous lymphokine preparations for characterization studies. These difficulties with regard to the study of lymphokines have caused workers in the field to experiment with cell lines and hybridomas.
The establishment of stable T cell lines secreting homogenous and distinct lymphokines has generated a large amount of information on the functions of soluble mediators [review by Moller (12)]. The continuous requirement for T cell growth factor (TCGF) for maintaining these clones has however hampered large-scale production of specific lymphokines. Recently, T-T hybridomas have been constructed (13-17) to immortalize lymphokine-secreting T cells. Since hybridomas are TCGF independent, some investigators in the field have thought they would serve as better sources than cloned T cell lines for preparing larger quantities of lymphokines for biochemical characterization.
Human lymphoid cell lines also produce a number of factors similar to mediators of cellular immunity released by lymphocytes. Gillis, et al., (18) showed that the human T cell lines Jurkat produces large quantities of human interleukin 2 (IL2) upon stimulation with mitogen. Farrar, et al., (19) showed that the mouse thymoma EL4 secretes high concentrations of mouse IL2 upon stimulation by the mutagen phorbol myristate acetate. These two cell lines produce such abundant quantities of the respective factors that detailed biochemical analysis of their function-structural relationships are underway.
Vesole, et al., (20) have described previous reports of lymphoid lines which secrete low levels of uncharacterized suppressor factors that inhibit DNA synthesis of mitogen stimulated lymphocytes, while noting that stimulation of lymphocyte growth (and thus of DNA synthesis) may also be caused by supernatants of lymphoid cell lines. In the Vesole reference itself, the authors indicate that the human lymphoid cell line CCRF-CEM ("CEM") secretes a substance which by itself increases DNA synthesis in lymphocytes. These authors also disclose that CEM secretes an uncharacterized substance which weakly inhibits mitogenic response in mononuclear cells stimulated with a mitogen such as PHA. From FIG. 1 in this reference, it can be seen that 50% inhibition of the mitogenic response is achieved at a dilution of about six-fold (i.e., about 10.sup.-0.8). This inhibiting substance is stated to elute on a Sephadex G-100 column with the albumin peak and thus has a molecular weight of about 70,000 dalton.
One of the references cited in Vesole (Yoshida, et al., J. Immunol., 117, 518 (1976)) also discloses work with CEM (among other cell lines) to examine its ability to release lymphokines into its culture medium, but there is no disclosure of any T cell suppressor factor being looked for or found.
Although various other mutants of various cell lines (including CEM) have been produced by other investigators, none of them even suggested that one could obtain a cell line secreting high titer suppressor factor.
Reference is made in this regard to Foung, et al., PNAS (U.S.A.), 79: 7484-7488 (December 1982), Lakow, et al., J. Immunol., 130, 169-172 (January 1983), and DeFreitas, et al. (16), who reported work with the human T cell line Jurkat.
Additionally, several authors have reported work with mutants of the T cell line CEM. Okada, et al., PNAS (U.S.A.) 78: 7717-7721 (December 1981), reported work with an 8-azaguanine-resistant mutant of CEM, while Irigoyen, et al., (17) reported establishment of a 6-thioguanine-resistant mutant from the cell line CEM.
Irigoyen, et al., describe the preparation of a 6-thioguanine (6T) resistant mutant of the CEM cell line and the subsequent fusing of this mutant with lectin-stimulated human T cells to produce human-human T cell hybridomas. No mention is made of the source of the parent CEM cell line. The method used for production of the mutant involved an initial mutation by ethyl methanesulfonate followed by exposure to increasing concentrations of 6T (1.25 .mu.g-5 .mu.g/ml), after which the mutant cells were maintained in 16.7 .mu.g/ml of 6-thioguanine.
Irigoyen, et al., is the only group known to Applicant which has reported on the characteristics of a 6T-resistant mutant CEM line. Their mutant line CEM-T15 (the only one discussed in the reference) was described as lacking the OKT3 antigen and having a chromosome number (karotype) of 35-47, the same as the parent CEM line used in its production. Additionally, Irigoyen's parent CEM line and the mutant line CEM-T15 both grew normally in the presence of 10.sup.-8 M aminopterin (0.04 .mu.g/ml), both died in the presence of 10.sup.-7 M aminopterin (0.04 .mu.g/ml), and there was differential growth (CEM grew, while CEM-T15 died) at 3.3.times.10.sup.-8 M and 6.6.times.10.sup.-8 M aminopterin (0.013 and 0.026 .mu.g/ml, respectively). The reference also indicates that CEM-T15 . . . "inhibits the background level of PFC (plaque forming cells) generated in cultures containing B cells and PWM (pokeweed mitogen)." This result indicates that CEM-T15 inhibits B cell differentiation. No information is given regarding any action on T cell differentiation.
These reports provided no suggestion that one could produce a stable mutant cell line which would have excellent growth characteristics and would secrete high titer suppressor factor. In fact, the present inventor observed in the course of the work leading to this invention that an azaguanine-resistant mutant CEM line was produced which showed deficiency in the enzyme hypoxanthine guanine ribotransferase (like the 6T-resistant mutant) but did not exhibit the beneficial characteristics found in the cell line of the present invention.